We wish to understand the partitioning of the eukaryotic genome into heterochromatin and euchromatin domains, determine the consequences of this differential packaging in terms of local chromatin structure and the capacity for gene expression, examine the process by which such differential packaging occurs during early embryogenesis, and analyze the roles of particular DNA and protein components in dictating these events. The results should contribute to an understanding of the abnormal regulation of genes that accompanies chromosome rearrangements. Four categories of studies have been planned: 1) Determine the functional significance of "C1A9", a heterochromatin-specific protein of Drosophila melanogaster which we have recently identified. The gene encoding C1A9 is being characterized, and its genetic locus is being mapped. We propose to isolate several conditional (temperature sensitive) lethal mutations in C1A9 to use in analysis of its role. 2) Identify additional heterochromatin-specific proteins. The immunocytological study of proteins which bind tightly to DNA, a characteristic of C1A9, will be continued. Further, we will use the monoclonal antibody to C1A9 to select heterochromatin fragments for biochemical analysis and carry out immunological screening of potential additional heterochromatin-specific proteins. 3) Examine the structural and functional consequences of placing a gene that is normally found in a euchromatic environment into a heterochromatic environment. First, we will use P-element transformation to place a heat shock protein gene (hsp 28) within the heterochromatic regions of the genome. Second, we will look at the effects on hsp 28 when it is flanked with sequences known to be major elements of heterochromatin (e.g. satellite DNA or type I rDNA inserts), but placed in a euchromatin region. Effects on both the local chromatin structure and the expression of hsp 28 will be examined. 4) Examine the process of heterochromatin formation during early development of the Drosophila embryo. Heterochromatin is absent from early cleavage nuclei in Drosophila, but present by the cellular blastoderm stage. Using DNA cleavage reagents, we will analyze the changes in chromatin structure that occur during early embryogenesis in genes that are known to be packaged as heterochromatin after cellular blastoderm. In parallel, we will use immunocytological techniques to study the time course of nuclear accumulation of the C1A9 antigen and other heterochromatin-specific proteins. These studies will provide a baseline description of heterochromatin formation against which the effects of various mutations (e.g. ts mutations of C1A9) can be tested.